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Abstract
Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.

Fig. 5. Localized expression of xSP-C and xSP-B in the developing lung. In situ hybridization performed on Xenopus embryos provides evidence that the expression of surfactant protein genes is localized to the lung. Whole mount in situ hybridization was performed using antisense RNA probes for xSP-C (A–C and E–F), Nkx2.1 (D), and xSP-B (G–I). (A) xSP-C is expressed in a broad crescent shape as viewed dorsally in a stage 41embryo. (B) xSP-C expression expands in the growing tubules of the lung at stage 44. (C) Lateral view of a stage 43embryo shows xSP-C expression just dorsal to the coiling gut tube. (E) Transverse section of a stage 41embryo shows xSP-C expression in the two lung tubes (arrows). (F) More anterior transverse section of the same embryo in (E) shows xSP-C expression in a single tube (arrow) before lung has split into two tubules. (H) SP-B is also expressed in a broad crescent shape as viewed dorsally in a stage 41embryo. (I) xSP-B expression expands in the growing tubules of the lung at stage 44. (G) Transverse section of a stage 39embryo shows expression of xSP-B in a widening tube (arrow) just anterior to the splitting of the two lung tubules. (D) Nkx2.1 is expressed in the forebrain (little arrow), thyroid (arrowhead), and lung (big arrow) at stage 36, preceding expression of xSP-C and xSP-B in the lung. Scale bar in (A) represents 0.5 mm in all panels.

Fig. 3. Expression of xSP-C and xSP-B at different developmental time points. RT-PCR was performed on cDNA from various developmental time points in order to determine when xSP-C and xSP-B genes were expressed in the developing embryo. The following cDNA was used in each lane: (1) water, (2) adult lung with no reverse transcriptase added to the RT reaction, (3) blank lane, (4) stage 2, (5) stage 6.5, (6) stage 10, (7) stage 15, (8) stage 21, (9) stage 24, (10) stage 27, (11) stage 31, (12) stage 33/34, (13) stage 35/35, (14) stage 41, (15) stage 43, (16) adult lung. xSP-C and ODC expression was detected after 35 cycles of PCR and xSP-B was detected after 30 cylces.

Fig. 4. Expression of xSP-C and xSP-B in adult tissues. Expression of the surfactant protein genes is limited to the lung in adult Xenopus laevis. RT-PCR was performed on cDNA from various adult tissues in order to determine where xSP-C and xSP-B genes were expressed in the adult frog. The following adult tissue cDNA was used in each marked lane: (H20) water, (-RT) lung with no reverse transcriptase added to the RT reaction, (Lu) lung, (St) stomach, (Li) liver, (He) heart, (Mu) skeletal muscle, and (In) intestine. xSP-C, SP-B, and ODC expression was detected after 30 cycles of PCR.